Vectors for transforming plants

ABSTRACT

Vectors for transforming plants with the use of  agrobacteria  which have been modified so as to elevate the possibility of the recognition of the border sequences of the vectors by vir proteins of the  agrobacteria , thereby lowering the possibility of the transfer of DNAs other than T-DNA into plant chromosomes. More particularly, the above-vectors are those to be used in transforming plants which have right and left border sequences which can be recognized by the vir proteins of the  agrobacteria , a T-DNA sequence which is located between these border sequences and into which a gene to be transferred into plants can be inserted, and a replication origin enabling the replication of the vectors in bacteria, characterized by having a plural number of left border sequences.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/JP00/05386 which has an Internationalfiling date of Sep. 30, 1999, which designated the United States ofAmerica and was not published in English.

TECHNICAL FIELD

The present invention relates to plant transformation vectors, moreparticularly, vectors useful in Agrobacterium-mediated planttransformation. The invention further relates to a method oftransforming plants using the vectors. The invention is particularlyuseful for generating transgenic plants that may be taken as food.

BACKGROUND ART

It has long been known that Agrobacterium (Agrobacterium tumefaciens),is a soil bacterium which causes Crown gall disease in manydicotyledonous plants. In the ninety-seventies, it was found that the Tiplasmid of Agrobacterium is involved in pathogenicity and that T-DNAwhich is part of the Ti plasmid is integrated into the plant genome. Itwas later revealed that the T-DNA contained the hormone synthesis genes(cytokinins and auxins) necessary for crown gall tumorigenesis and thatthose genes, although derived from bacteria, are expressed in plants. Agroup of genes that are located in the virulence region (Vir region) ofthe Ti plasmid are necessary to the excision of T-DNA and its transferto plants, and furthermore, the border sequences that are located onopposite ends of T-DNA are required for the excision, which are calledthe right border sequence and the left border sequence. Agrobacteriumrhizogenes, another Agrobacterium species has a similar system involvingthe Ri plasmid.

Stated more specifically, the proteins produced on the basis of thegenes located in the vir region (vir proteins) recognize the right andleft border sequences to integrate the T-DNA located between the bordersequences into plant genome. This function provided the basis for thetransformation of plants with a foreign gene pre-inserted into T-DNA,thereby giving rise to the development of Agrobacterium-mediated planttransformation technology.

Most recently, however, several reports have appeared describing that,in certain kinds of plants, it is sometimes observed that T-DNA is notexcised at the border sequences, and hence, T-DNA can be transferredinto the plant chromosome together with a region adjacent to T-DNA(Ramanathan et al., Plant Molecular Biology 28, 1149–1154 (1995), andKononov et al., Plant Journal 11, 945–957 (1997)). If a DNA elementother than T-DNA is co-transferred, the resulting transgenic plants willbe suspected of having unexpected characteristics, which could have anegative impact on public acceptance of food products made of transgenicplants. It is therefore desired to develop a method whereby it can beensured that unnecessary non-T-DNA sequences of Agrobacterium will nottransfer to plant chromosomes.

The inventors supposed that the vir proteins of Agrobacterium sometimesfail to recognize the border sequences and this may explain the reasonwhy non-T-DNA is transferred into plant chromosome together with T-DNA.No vectors have yet been developed that can suppress or reduce thetransfer of non-T-DNA segment with a view to solving said problem.

Based on the above supposition, the inventors have conducted intensivestudies for creating vectors for use in Agrobacterium-mediatedtransformation. In order to reduce the probability that non-T-DNAelement is transferred to plant chromosome the inventors modified thevector with a view to increasing the efficiency of the vir proteins ofAgrobacterium to recognize the border sequence/s. As a result, it hasbeen found that while two border sequences exist in the transformationvector, the probability of the integration of non-T-DNAs can be reducedby providing a plurality of left border sequences. The present inventionhas been accomplished on the basis of this finding.

SUMMARY OF THE INVENTION

The present invention provides a plant transformation vector based onthe function of Agrobacterium, wherein the left border sequence has beenmodified such as to reduce the possibility of the integration of anynon-T-DNA segment into plant chromosomes. More particularly, theinvention provides a plant transformation vector comprising a rightborder sequence and a left border sequence that can be recognized by thevir proteins of Agrobacterium, a T-DNA region located between theseborder sequences and into which a gene to be introduced into the plantcan be inserted, and a replication origin (ori) that enables replicationof said vector in bacteria (e.g. Agrobacterium and bacteria for vectoramplification), wherein said left border sequence has been modified suchas to reduce the possibility of integration of any non-T-DNA sequenceinto plant chromosomes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows vector pSLB0 used in the Example; this vector was preparedfrom pSB11 by modifying it to have within the T-DNA region a cassette,for expressing the hygromycin resistance gene under control of aubiquitin promoter and a ubiquitin intron, then inserting a cassette forexpressing the GUS gene containing a catalase intron under control of aubiquitin promoter, at the site recognizable by restriction enzyme StuI;and

FIG. 2 shows maps of the areas of vectors pSLB0, pSLB2 and pSLB3 in theneighborhood of the left border sequence (which is hereunder sometimesreferred to as LB). To prepare pSLB2 and pSLB3, a synthetic DNA fragmenthaving two or three left border sequences was inserted into pSLB0between LB and the GUS expressing cassette at the restriction site ofPvuII. In pSLB2, two left border sequences were inserted by thesynthetic DNA fragment (the inserted left border sequence/s is hereunderreferred to as sLB) to give a total of three LBs, and in pSLB3, threesLBs were inserted by the synthetic DNA fragment to give a total of fourLBs.

DETAILED DESCRIPTION OF THE INVENTION

(1) Vector Preparation

The vector of the invention has the left border sequence modified tooperate such as to reduce the possibility of the integration of anunnecessary DNA sequence/s, i.e. a non-T-DNA sequence/s into plantchromosomes. In a preferred embodiment, the invention provides thevectors having the left border sequence modified such as to comprise theplacement of more than one DNA sequence that can be recognized by virproteins (e.g. a known left border sequence).

DNA fragments comprising more than one left border sequence can beprepared by various known methods on the basis of known bordersequences. For example, one can synthesize a single-stranded DNAmolecule having the same sequence as a left border sequence which iscontained in an available Ti plasmid, prepare the double-stranded DNAmolecule from the single strand, and if necessary, link two or more ofsuch double-stranded DNAs together. The obtained DNA fragment can bethen inserted into a plant transformation vector at a suitablerestriction site nearby up/downstream of the existing left bordersequence located downstream of the T-DNA region. In this way, the vectorof the invention can be easily constructed.

The plant transformation vector from which the vector of the inventioncan be prepared by modifying its left border sequence should at leasthave a right and a left border sequences that can be recognized by virproteins, a T-DNA sequence located between the right and left bordersequences and into which can be inserted a gene to be introduced intothe plant, and a replication origin that can operate in bacteria forreplication of the vector (e.g. Escherichia coli). The preferred planttransformation vector has a replication origin that can operate inAgrobacterium.

As long as these requirements are satisfied, various vectors can bemodified on the left border sequence. For example, the various vectorsused in the following plant transformation methods based onAgrobacterium can be modified:

-   -   (i) a small intermediate cloning vector having right and left        border sequences and which has a foreign gene inserted into        T-DNA, and an acceptor Ti plasmid having the vir region are        subjected to homologous recombination to prepare a hybrid Ti        plasmid vector, and the plant is infected with Agrobacterium        containing the hybrid Ti plasmid vector;    -   (ii) a foreign gene is inserted into the T-DNA region of a small        Ti plasmid having no vir region (the plasmid is commonly called        a mini plasmid or a micro Ti plasmid and is capable of        replication in many bacteria) and the plasmid is introduced into        Agrobacterium which harbors a plasmid having the vir region but        no T-DNA, and the plant is infected with the Agrobacterium        containing the two plasmids;    -   (iii) a small intermediate cloning vector having a right and a        left border sequences and which has an foreign gene inserted        into T-DNA, and an acceptor Ti plasmid having a portion of the        vir region (i.e., vir gene lacking a portion of the full length        of the vir region) are subjected to homologous recombination to        prepare a hybrid Ti plasmid vector, the hybrid Ti plasmid vector        is introduced into Agrobacterium which has the vir region (full        length) harboring but T-DNA deficient plasmid introduced in it,        and the plant is infected with the Agrobacterium containing the        two plasmids. The various vectors used in these methods can be        modified on the left border sequence. Small Ti plasmids with a        modification on the left border sequence are easy to handle in        operations such as for modifying the foreign gene in T-DNA and        hence are a preferred embodiment of the vector of the invention.        Examples of such small Ti plasmids include pBI101 and pBI121        (both being available from CLONTECH), as well as pSB11 which was        used in the Example to be described later.

The concept of the invention is applicable not only to the Ti plasmidbut also to the Ri plasmid.

The vector of the invention may contain a marker gene in the T-DNAsequence that permits selection of the transformant, such as anantibiotic resistance gene or a luminescence gene. To be specific,commonly used marker genes may be employed in the usual manner and theyinclude antibiotic resistance genes such as those conferring resistanceto tetracycline, ampicillin, kanamycin, neomycin, hygromycin andspectinomycin, and luminescence genes such as the luciferase gene,β-galactosidase, green fluorescence protein (GFP), β-lactamase andchloramphenicol acetyl transferase (CAT) genes. Besides these genes, thevector may contain another marker gene outside of the T-DNA sequence,preferably downstream of the left border sequences. The marker geneplaced in that position is useful for evaluating the effectiveness ofthe modified border sequences.

The term “replication origin” as used in this specification means aspecific DNA region in which the replication reaction is initiated,commonly called Ori.

(2) Transformation

To use the vector of the invention, a foreign gene for the intendedtransformation is inserted into the T-DNA region. The foreign gene to beinserted usually contains a promoter that can operate in the host plantand the structural gene encoding the characteristic to be conferred tothe plant linked downstream of the promoter. If necessary, more than onegene may be linked together and, in addition or alternatively, asequence for enhancing the efficiency of expression may be interposedbetween the promoter and the downstream structural gene before insertioninto the T-DNA region.

Before being introduced into a target plant, the vector of the inventionwhich harbors the foreign gene is introduced into a bacterial ofAgrobacterium species capable of infecting the plant (e.g. Agrobacteriumtumefaciens). To this end, various methods well known to the skilledartisan can be employed. For example, the vector may be transferred intothe Agrobacterium by conjugation; if possible, the Agrobacterium may bedirectly transformed with the vector of the invention containing theforeign gene.

Conventional techniques may be employed to infect the plant with theAgrobacterium containing the vector of the invention and they include,for example, wounding part of the plant body and infecting it with thebacterium, infecting the callus with the bacterium, co-cultivating theprotoplast and the bacterium, and co-cultivating slices of the leaftissue together with the bacterium. The transformed cells obtained bythese methods can be selected by using the suitable selection marker/sor assaying if they express the intended characteristic. The transformedcells may further be differentiated by the prior art technology to yielda recombinant plant body.

In order to integrate that T-DNA containing the foreign gene into thechromosomal DNA in the plant, the vir region is necessary. The virregion may be supplied from the vector having the foreign gene or from adifferent vector.

The plant cells transformed with the vector of the invention may bedifferentiated by the prior art technology to yield a recombinant plantbody. The transformed plant may be selected by using a suitableselection marker or assaying if it expresses the intendedcharacteristic.

Whether a DNA sequence/s unnecessary for the intended transformation hasbeen integrated into plant chromosomes or not can be determined byvarious methods well known to the skilled artisan. For example,oligonucleotide primers are synthesized on the basis of the vector DNAsequence/s outside the borders and with these primers, PCR is performedto analyze the chromosomal DNA sequences in the transformed plant. Inthe case of transformation with a vector that contains a marker geneoutside the T-DNA sequence, analysis can be done by assaying if themarker gene has been expressed.

The vector of the invention is characterized by its function to reducethe possibility of the integration of a DNA sequence/s unnecessary forthe intended transformation. The term “to reduce the possibility of theintegration” means that, compared to the use of a vector which is notmodified on the left border sequence, the frequency of the integrationof the unnecessary DNA sequence/s into host chromosomes is low, or thelength of the integrated unnecessary sequence is short, or there is nosuch integration and; in addition or alternatively, compared to the useof a vector which is not modified on the left border sequence, thefrequency of unintended transformation is low, or unintendedtransformation is slight, or there is no occurrence of suchtransformation. The term “DNA sequence unnecessary for the intendedtransformation” means a portion or fragment of the DNA sequence locatedoutside the T-DNA sequence in the vector (namely, non-T-DNA). It doesnot matter whether it is functional by itself or encodes a polypeptideor protein.

Various plants can be transformed by the transforming method of theinvention and they include monocotyledonous plants such as maize,sorghum, triticale, barley, oats, rye, wheat, onion and rice, anddicotyledonous plants such as soybean, alfalfa, tobacco, rape,sunflower, potato, pepper and tomato. The method of the invention canreduce the possibility of the integration of a DNA sequence/sunnecessary for the intended transformation into plant chromosomes andthe transgenic plants obtained by using the transformation method areless likely to have unexpected characteristics. Therefore, the method ofthe invention is suitable for transforming plants that can be taken asfood by other organisms and regarding which there is particular concernabout the possibility that the non-T-DNA sequence/s will transfer intoplant chromosomes by Agrobacterium-mediated transformation. The methodis most suitable for transforming monocotyledonous plants, inparticular, rice.

Unless otherwise noted, the term “plant or plants” as used in thespecification covers not only a plant body (individual) but also itsseed (germinated or immature), part (leaf, root, stem, flower, stamen,pistil or slices of these), culture cell, callus and protoplast.

EXAMPLE Example 1

(1) Preparing Vectors

Plasmid vector pSB11 (Genbank Accession No. AB027256, Komari et al.,Plant Journal 10, 165–174 (1966)) was modified to have within the T-DNAregion a cassette for expressing the hygromycin resistance gene (HPT) bymeans of a ubiquitin promoter and a ubiquitin intron (Christensen etal., Plant Molecular Biology 18, 675–689 (1992) and into the plasmid, acassette for expressing a catalase intron containing GUS gene (Ohta etal., Plant Cell Physiology 31, 805–813 (1990)) by means of a ubiquitinpromoter was inserted at the site recognizable by restriction enzymeStuI. The thus prepared plasmid was designated pSLB0 (see FIG. 1 and SEQID:NO. 1). The nucleotide sequence of pSLB0 is shown as SEQ ID:NO. 1.

Then, based on the nucleotide sequence of Ti plasmid pTiAch5 (GenbankAccession No. K00548), a synthetic DNA containing a left border sequence(hereunder abbreviated as LB) and the complementary synthetic DNA wereprepared, they were annealed and processed to form blunt ends, and thenused to prepare DNA fragments respectively having two and three LBsequences. The nucleotide sequences of the two synthetic DNAs are shownas SEQ ID:NO. 2 and SEQ ID:NO. 3. Each of the DNA fragments was insertedinto pSLB0 between LB and the GUS expressing cassette at the siterecognized by restriction enzyme PvuII; in this way, vectors wereprepared that had more than one LB attached. The vector having two ofthe LB introduced by the synthetic DNA (which is hereunder sometimesreferred to as sLB) to give a total of three LBs was designated pSLB2,and the vector having three sLBs introduced to give a total of four LBswas designated pSLB3 (for the maps of areas of pSLB0, pSLB2 and pSLB3 inthe neighborhood of the synthetic LBs, see FIG. 2). Each of these threeplasmids was introduced into Agrobacterium tumefaciens LBA4404 whichalready had plasmid vector pSB1 introduced (Genbank Accession No.AB027255, Komari et al., Plant Journal 10, 165–174 (1996)). They weresubjected to the following tests.

(2) Transformation

Calli derived from the immature embryo of rice variety “Asanohikari”were transformed with LBA4404(pSLB0), LBA4404(pSLB2) and LBA4404(pSLB3)in accordance with the method of Hiei et al. (Hiei et al., Plant Journal6, 271–282 (1994)).

(3) Analysis of the Expression of GUS Gene in Transformants

Some leaves of the hygromycin-resistant plants obtained in Example 2were stained with X-Gluc to check for the expression of the GUS gene.Seventeen out of the 340 plant individuals transformed withLBA4404(pSLB0) expressed the GUS gene, indicating that Agrobacteriumderived DNA outside the border sequences was introduced into 5% of theplant individuals transformed with the conventional vector having onlyone LB. On the other hand, the number of plant individuals transformedwith LBA4404(pSLB2) and LBA4404(pSLB3) and which expressed the GUS genedecreased with the increasing number of synthetic LBs (Table 1). Thisindicates that the integration of synthetic LBs into the vectordecreased the likelihood for DNA beyond the left border sequence to betransferred to the plant.

(4) Analysis of Genomic DNA in the Individuals not Expressing the GUSGene

In some of the individuals that did not express the GUS gene in (3), DNAbeyond the left border sequence may have been introduced into plantchromosomes but not far enough to the ubiquitin promoter for triggeringthe expression of the GUS gene. To verify this possibility in each groupof plants that did not express the GUS gene, about 60 independenttransformants were randomly chosen and genomic DNA was extracted andsubjected to PCR analysis. The primers used in PCR analysis were soprepared as to permit amplification of the region extending from alocation between the inherent LB and the synthetic LB to a location inthe GUS gene. The sequences of the primers are shown as SEQ ID:NO. 4 andSEQ ID:NO. 5.

As a result of the PCR analysis, seven out of the 67 plant individuals(10.4%) transformed with LBA4404(pSLB0) showed DNA amplification,revealing that when the conventional vector having only one LB was used,Agrobacterium derived DNA other than the desired T-DNA was integratedinto chromosomes in the created transformants with a frequency of 10.4%of higher. In contrast, DNA amplification was found to take place innone of the plant individuals transformed with LBA4404(pSLB2) andLBA4404(pSLB3) that had synthetic LBs.

The results are shown in Tables 1 and 2.

TABLE 1 Analysis for the Expression of GUS Gene Vector Number oftransformants Percentage of GUS expressing plants pSLB0 340 5.0 pSLB2327 1.2 pSLB3 370 0.8

TABLE 2 Analysis of Genomic DNA No. of plants from No. of plants inwhich Vector which DNA was extracted DNA was amplified by PCR pSLB0 67 7pSLB2 55 0 pSLB3 58 0

As can be seen from the above, the present invention decreased theintegration of a DNA sequence/s outside the border sequences in plantchromosomes and made it possible to increase the efficiency ofintroducing only the intended T-DNA.

The foregoing description of the invention concerns primarily the use oftwo or more left border sequences, which may be derived from the same ordifferent species of Agrobacterium. It should, however, be stressed thatthe fundamental concept of the invention lies in modifying the leftborder sequence in plant transformation vectors such that it can berecognized by vir proteins more efficiently to reduce the integration ofany unnecessary non-T-DNA sequence to plant chromosomes. Therefore, thepresent invention embraces all vectors that have the modified leftborder sequence/s capable of achieving the same result. In addition tothe examples described above, the modified left border sequences includethe following: (1) those sequences which are derived from the sequencealready existing in relevant plasmids by deletion, substitution oraddition of one or more nucleotides in the existing left border sequenceto be recognized by vir proteins more efficiently; (2) those sequenceswhich are derived from the sequence already existing in the plasmid bydeletion, substitution or addition of one or more bases in any sequencenear the existing left border sequence to be recognized by vir proteinsmore efficiently; (3) those sequences which contain a plurality of anysequences that can be recognized by vir proteins; and (4) anycombinations of (1)–(3).

1. A vector for Agrobacterium-mediated plant transformation comprising:a T-DNA right border region that is recognized by the vir proteins ofAgrobacterium; a T-DNA left border region comprising at least two T-DNAleft border sequences that is recognized by the vir proteins ofAgrobacterium; a T-DNA region located between these border regions andinto which a nucleotide sequence to be introduced into the plant can beinserted; and a replication origin that enables replication of saidvector in bacteria; wherein said vector when used in theAgrobacterium-mediated plant transformation reduces the integrationfrequency of a non-T-DNA segment into a plant chromosome, as comparedwith a vector comprising a T-DNA left border region consisting of asingle T-DNA left border sequence.
 2. The vector according to claim 1,wherein the T-DNA region contains a marker comprising a polynucleotidesequence that permits the selection of a plant transformed with thevector.
 3. The vector according to claim 1, wherein the replicationorigin permits replication of the vector in a bacterial cell for vectoramplification and an Agrobacterium host cell.
 4. A method fortransforming a plant cell comprising the steps of: introducing thevector according to any one of claims 1, 2 or 3 into an Agrobacteriumhost cell; and transforming a plant cell with the Agrobacterium hostcell harboring the vector, thus obtaining a transformed plant cell.
 5. Aplant transformed by the method of claim
 4. 6. A method for reducing theintegration frequency of non-T-DNA segment of a vector forAgrobacterium-mediated plant transformation, comprising the steps of:introducing the vector according to any one of claims 1, 2 or 3 into anAgrobacterium host cell; and transforming a plant cell with theAgrobacterium host cell harboring the vector, thus obtaining atransformed plant cell, wherein the integration frequency of non-T-DNAsegment into the chromosome of the plant cell is reduced as compared tothe case when a vector comprising a T-DNA left border region consistingof a single T-DNA left border sequence is used.
 7. The vector accordingto claim 1, wherein the T-DNA left border region comprises at leastthree T-DNA left border sequences.
 8. A method for transforming a plantcomprising the steps of: introducing the vector according to any one ofclaims 1, 2 or 3 into an Agrobacterium host cell; and transforming aplant by infecting the plant with the Agrobacterium host cell harboringthe vector; thus obtaining a transformed plant.